All three isoforms of nitric oxide synthase (NOS) require tetrahydrobiopterin (BH4) as a cofactor. However, the precise mechanism by which BH4 enhances the catalytic activity of NOS remains unknown. It has been proposed that BH4 acts as an electron donor in the monoxygenation of L-arginine or as a stabilizing agent for subunit dimerization. More recently, BH4 was shown to prevent NOS from feedback inhibition of nitric oxide. This effect was attributed to autoxidation of BH4, which forms superoxide that subsequently reacts with nitric oxide to form peroxynitrite. Regardless of the mechanism of protection, the fact that BH4 undergoes autoxidation to form superoxide is of considerable significance, as BH4 is routinely added at various concentrations in the assay used to determine NOS activity. In this study, we have used both 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) to monitor radical formation from autoxidation of BH4. The rationale for using DEPMPO is that, unlike DMPO/ OOH which spontaneously decays to DMPO/ OH, DEPMPO/ OOH does not spontaneously decay to DEPMPO/ OH. We have investigated the effect of spermine NONOate (SNN), a slow and spontaneous nitric oxide donor, on the formation of DEPMPO adducts.